title: Identification of residues essential for the activity and substrate affinity of L-carnitine dehydrogenase.
abstract: Recently, two L-carnitine dehydrogenases from soil isolates Rhizobium sp. (Rs-CDH) and Xanthomonas translucens (Xt-CDH) have demonstrated to exhibit mutually differing affinities toward L-carnitine. To identify residues important for affinity to the substrate, we compared the primary structure of Xt-CDH and Rs-CDH with the recognized 3D structure of 3-hydroxyacyl-CoA dehydrogenase (PDB code: 1F0Y). Then, six residues of Xt-CDH (Phe143, Gly188, Ile190, Ala191, Gly223, and Ala224) and the corresponding residues of Rs-CDH (Tyr140, Ala185, Val187, Gly188, Ser220, and Phe221) were selected for further mutagenesis. The residues of Xt-CDH were replaced with that of Rs-CDH at the corresponding position and vice versa. All Rs-CDH mutants exhibited slight effects on substrate affinity, except for the double mutants Rs-V187I/G188A, which was devoid of enzyme activity. All Xt-CDH mutants showed different K m values. Xt-F143Y caused a higher increase in the K m value. Furthermore, the kinetic parameters of 10 mutants at Xt-F143 and Rs-Y140 were investigated. All Rs-Y140 mutants, except aromatic residues (Phe, Trp), produced proteins that were almost entirely devoid of enzyme activity and with disrupted affinity to L-carnitine. All Xt-F143 variants showed a marked reduction (P ≤ 0.05) in enzyme activity. Overall, our results suggest that the aromatic rings of Tyr140 in Rs-CDH and Phe143 of Xt-CDH are essential for substrate recognition.
authors.affiliation: United Graduate School of Agricultural Sciences, Tottori University, Koyama, Tottori, 680-8553, Japan.
authors.forename: Mohamed M
authors.initials: MM
authors.lastname: Eltayeb
authors.forename: Isam A
authors.initials: IA
authors.lastname: Mohamed Ahmed
authors.forename: Jiro
authors.initials: J
authors.lastname: Arima
authors.forename: Nobuhiro
authors.initials: N
authors.lastname: Mori
mesh_terms: D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002331:Carnitine / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000378:metabolism; D011487:Protein Conformation; D012231:Rhizobium / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D014443:Tyrosine / Q000378:metabolism; D014974:Xanthomonas / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D014443:Tyrosine; D010649:Phenylalanine; D000429:Alcohol Oxidoreductases; C019842:carnitine dehydrogenase; D002331:Carnitine